The overall goal of this application is to understand biochemical mechanisms that underlie the function of rhesus monkey TRIM5 (rhTRIM5). rhTRIM5 blocks HIV-1 infection by acting at two or more post-entry stages. Importantly, rhTRIM5 E3 ubiquitin ligase activity and functional proteasome are important for its full restriction activity. According to the current model of rhTRIM5 function, rhTRIM5 recognizes and binds the incoming restricted virus capsid. This event triggers redistribution of rhTRIM5 molecules to the vicinity of the capsid leading to its accelerated uncoating, and engages the ubiquitin proteasome machinery that mediates capsid degradation. Interestingly, in the cell, rhTRIM5 exhibits a complex behavior pattern characterized by rapid turnover rate and constant exchange of rhTRIM5 molecules between different discrete cytoplasmic compartments. These processes are probably tightly regulated, even in the absence of virus infection, and could be important for rhTRIM5's ability to restrict retrovirus infection. To gain insight into the molecular mechanisms by which rhTRIM5 restricts retrovirus infection, we purified cellular proteins that are bound to rhTRIM5 in intact cells and identified them by a shotgun proteomic approach. Importantly, we found that a significant fraction of rhTRIM5 is specifically associated with 26S proteasome. Thus, we hypothesize that rhTRIM5 binding to 26S proteasome is crucial for the early post-entry infection block to occur. Interestingly, we also identified additional components of ubiquitin proteasome system that specifically associate with rhTRIM5, such as subunits of E3 ubiquitin ligases. These enzymes are likely regulators of rhTRIM5 levels, because rhTRIM5 turnover rate is regulated by polyubiquitylation. In this application we propose to scrutinize the roles of rhTRIM5 binding with 26S proteasome and with other specific components of the ubiquitin proteasome system, for rhTRIM5-mediated restriction and rhTRIM5 turnover rate. Subsequently, we plan to initiate structural studies of selected molecular interactions that are crucial mediators of these processes. Ultimately, our studies will provide a biochemical/molecular framework for understanding the mechanism(s) by which rhTRIM5 blocks HIV-1 infection, and how these mechanism(s) are regulated. This knowledge could lead to the identification of new targets in viral life cycle for development of antivirals and to the conception of new strategies to control HIV infection. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding molecular mechanism that TRIM5 uses to defends cells from infection by HIV-1. Knowledge gained from our studies could lead to the conception of new strategies to control HIV infection and to the identification of new targets for development of anti-retroviral drugs.